Amplification and detection of shigella spp. and enteroinvasive strains of Escherichia coli

ABSTRACT

Amplification primers and methods for specific amplification and detection of a Shigella spp. and enteroinvasive stains of  Escherichia coli  (EIEC) target are disclosed. The primer-target binding sequences are useful for amplification and detection of Shigella and EIEC target in a variety of amplification and detection reactions.

This is a division of U.S. patent application Ser. No. 09/289,750, filedApr. 12, 1999, U.S. Pat. No. 6,060,252.

FIELD OF THE INVENTION

The present invention relates to methods for determining the presence orabsence of Shigella spp. and enteroinvasive strains of Escherichia coli(EIEC) in patients, food or water. The method involves using nucleicacid primers to amplify specifically a Shigella and EIEC ipaH target,preferably using one of the techniques of Strand DisplacementAmplification (SDA), thermophilic Strand Displacement Amplification(tSDA) or fluorescent real time tSDA, and optionally using amicroelectronic array.

BACKGROUND OF THE INVENTION

Organisms of the genus Shigella cause classic bacillary dysentery thatis characterized by severe diarrhea and abdominal pain. Shigella spp.are typically associated with self-limiting infections that are rarelyfatal except in children or the elderly. However, infection with S.dysenteriae can cause a severe form of the disease with up to 20% ofcases proving fatal. Like Shigella spp., EIEC causes enteritis withfever, abdominal cramps and watery diarrhea. Strains of EIEC are,however, considered to be rare both in the developed and developingworld. Nucleic acid amplification is a powerful technology thatfacilitates rapid detection of specific target sequences. It istherefore a promising technology for the rapid detection andidentification of Shigella spp. and EIEC. The oligonucleotide primers ofthe present invention are applicable to the amplification and detectionof Shigella- and EIEC-specific regions of the ipaH virulence gene. TheipaH gene is approximately 16 kb in length and is found in multiplecopies on both the large virulence-associated plasmid of Shigella spp.and the bacterial chromosome.

The following terms are defined herein as follows:

An amplification primer is a primer for amplification of a targetsequence by extension of the primer after hybridization to the targetsequence. Amplification primers are typically about 10-75 nucleotides inlength, preferably about 15-50 nucleotides in length. The total lengthof an amplification primer for SDA is typically about 25-50 nucleotides.The 3′ end of an SDA amplification primer (the target binding sequence)hybridizes at the 3′ end of the target sequence. The target bindingsequence is about 10-25 nucleotides in length and confers hybridizationspecificity on the amplification primer. The SDA amplification primerfurther comprises a recognition site for a restriction endonuclease 5′to the target binding sequence. The recognition site is for arestriction endonuclease which will nick one strand of a DNA duplex whenthe recognition site is hemimodified, as described by G. Walker, et al.(1992. Proc. Natl. Acad. Sci. USA 89:392-396 and 1992 Nucl. Acids Res.20:1691-1696). The nucleotides 5′ to the restriction endonucleaserecognition site (the “tail”) function as a polymerase repriming sitewhen the remainder of the amplification primer is nicked and displacedduring SDA. The repriming function of the tail nucleotides sustains theSDA reaction and allows synthesis of multiple amplicons from a singletarget molecule. The tail is typically about 10-25 nucleotides inlength. Its length and sequence are generally not critical and can beroutinely selected and modified. As the target binding sequence is theportion of a primer which determines its target-specificity, foramplification methods which do not require specialized sequences at theends of the target the amplification primer generally consistsessentially of only the target binding sequence. For example,amplification of a target sequence according to the invention using thePolymerase Chain Reaction (PCR) will employ amplification primersconsisting of the target binding sequences of the amplification primersdescribed herein. For amplification methods that require specializedsequences appended to the target other than the nickable restrictionendonuclease recognition site and the tail of SDA (e.g., an RNApolymerase promoter for Self-Sustained Sequence Replication (3SR),Nucleic Acid Sequence-Based Amplification (NASBA) or theTranscription-Based Amplification System (TAS)), the requiredspecialized sequence may be linked to the target binding sequence usingroutine methods for preparation of oligonucleotides without altering thehybridization specificity of the primer.

A bumper primer or external primer is a primer used to displace primerextension products in isothermal amplification reactions. The bumperprimer anneals to a target sequence upstream of the amplification primersuch that extension of the bumper primer displaces the downstreamamplification primer and its extension product.

The terms target or target sequence refer to nucleic acid sequences tobe amplified. These include the original nucleic acid sequence to beamplified, the complementary second strand of the original nucleic acidsequence to be amplified and either strand of a copy of the originalsequence which is produced by the amplification reaction. These copiesserve as amplifiable targets by virtue of the fact that they containcopies of the sequence to which the amplification primers hybridize.

Copies of the target sequence which are generated during theamplification reaction are referred to as amplification products,amplimers or amplicons.

The term extension product refers to the copy of a target sequenceproduced by hybridization of a primer and extension of the primer bypolymerase using the target sequence as a template.

The term species-specific refers to detection, amplification oroligonucleotide hybridization to a species of organism or a group ofrelated species without substantial detection, amplification oroligonucleotide hybridization to other species of the same genus orspecies of a different genus.

The term assay probe refers to any oligonucleotide used to facilitatedetection or identification of a nucleic acid. Detector probes, detectorprimers, capture probes, signal primers and reporter probes as describedbelow are examples of assay probes.

The term amplicon refers to the product of the amplification reactiongenerated through the extension of either or both of a pair ofamplification primers. An amplicon may contain exponentially amplifiednucleic acids if both primers utilized hybridize to a target sequence.Alternatively, amplicons may be generated by linear amplification if oneof the primers utilized does not hybridize to the target sequence. Thus,this term is used generically herein and does not imply the presence ofexponentially amplified nucleic acids.

A microelectronic array (or electronic microarray) is a device with anarray of electronically self-addressable microscopic locations. Eachmicroscopic location contains an underlying working direct current (DC)micro-electrode supported by a substrate. The surface of each microlocation has a permeation layer for the free transport of smallcounter-ions, and an attachment layer for the covalent coupling ofspecific binding entities.

An array or matrix is an arrangement of locations on the device. Thelocations can be arranged in two dimensional arrays, three dimensionalarrays, or other matrix formats. The number of locations can range fromseveral to at least hundreds of thousands.

Electronic addressing (or targeting) is the placement of chargedmolecules at specific test sites. Since DNA has a strong negativecharge, it can be electronically moved to an area of positive charge. Atest site or a row of test sites on the microchip is electronicallyactivated with a positive charge. A solution of DNA probes is introducedonto the microchip. The negatively charged probes rapidly move to thepositively charged sites, where they concentrate and are chemicallybound to that site. The microchip is then washed and another solution ofdistinct DNA probes can be added. Site by site, row by row, an array ofspecifically bound DNA probes can be assembled or addressed on themicrochip. With the ability to electronically address capture probes tospecific sites, the system allows the production of custom arraysthrough the placement of specific capture probes on a microchip. In thisconnection, the term “electronically addressable” refers to a capacityof a microchip to direct materials such as nucleic acids and enzymes andother amplification components from one position to another on themicrochip by electronic biasing of the capture sites of the chip.“Electronic biasing” is intended to mean that the electronic charge at acapture site or another position on the microchip may be manipulatedbetween a net positive and a net negative charge so that molecules insolution and in contact with the microchip may be directed toward oraway from one position on the microchip or form one position to another.

Electronic concentration and hybridization uses electronics to move andconcentrate target molecules to one or more test sites (or capturesites) on the microchip. The electronic concentration of sample DNA ateach test site promotes rapid hybridization of sample DNA withcomplementary capture probes. In contrast to the passive hybridizationprocess, the electronic concentration process has the distinct advantageof significantly accelerating the rate of hybridization. To remove anyunbound or nonspecifically bound DNA from each site, the polarity orcharge of the site is reversed to negative, thereby forcing any unboundor nonspecifically bound DNA back into solution away from the captureprobes. In addition, since the test molecules are electronicallyconcentrated over the test site, a lower concentration of target DNAmolecules is required, thus reducing the time and labor otherwiserequired for pre-test sample preparation. The term “capture site” refersto a specific position on an electronically addressable microchipwherein electronic biasing is initiated and where molecules such asnucleic acid probes and target molecules are attracted or addressed bysuch biasing.

Electronic stringency control is the reversal of electrical potential toremove unbound and nonspecifically bound DNA quickly and easily as partof the hybridization process. Electronic stringency provides qualitycontrol for the hybridization process and ensures that any bound pairsof DNA are truly complementary. The precision, control, and accuracy ofplatform technology, through the use of the controlled delivery ofcurrent in the electronic stringency process, permits the detection ofsingle point mutations, single base pair mismatches, or other geneticmutations, which may have significant implications in a number ofdiagnostic and research applications. Electronic stringency is achievedwithout the cumbersome processing and handling otherwise required toachieve the same results through conventional methods. In contrast topassive arrays, this technology can accommodate both short and longsingle-stranded fragments of DNA. The use of longer probes increases thecertainty that the DNA which hybridizes with the capture probe is thecorrect target. Electronic stringency control reduces the requirednumber of probes and therefore test sites on the microchip, relative toconventional DNA arrays. In contrast, traditional passive hybridizationprocesses are difficult to control and require more replicants of everypossible base pair match so that correct matches can be positivelyidentified.

Electronic multiplexing allows the simultaneous analysis of multipletests from a single sample. Electronic multiplexing is facilitated bythe ability to control individual test sites independently (foraddressing of capture probes or capture molecules and concentration oftest sample molecules) which allows for the simultaneous use ofbiochemically unrelated molecules on the same microchip. Sites on aconventional DNA array cannot be individually controlled, and thereforethe same process steps must be performed on the entire array. The use ofelectronics in this technology provides increased versatility andflexibility over such conventional methods.

SUMMARY OF THE INVENTION

The present invention provides oligonucleotide primers that can be usedfor amplification of target sequences found in Shigella spp. and EIEC.More specifically, the target sequence comprises segments of the ipaHgene. The amplification primers have been designed for high-efficiency,high-specificity amplification at elevated temperatures, such as in tSDAand the PCR, however, they are also useful in lower-temperatureamplification reactions such as conventional SDA, 3SR or NASBA.Oligonucleotide assay probes that hybridize to the assay region of theamplified target are used to detect the amplification products.

The oligonucleotides of the invention may be used after culture as ameans for confirming the identity of the cultured organism.Alternatively, they may be used with clinical samples from humans oranimals, such as fecal material or urine, or with samples ofcontaminated food or water for detection and identification of Shigellaspp.s' and EIECs' nucleic acid using known amplification methods. Ineither case, the inventive oligonucleotides and assay methods provide ameans for rapidly discriminating between Shigella spp. and EIEC andother microorganisms, allowing the practitioner to identify thismicroorganism rapidly without resorting to the more traditionalprocedures customarily relied upon. Such rapid identification of thespecific etiological agent involved in an infection provides informationthat can be used to determine appropriate action within a short periodof time.

SUMMARY OF THE SEQUENCES

SEQ ID NOs:1-3 are sequences of oligonucleotides used as upstreamprimers for amplification of the ipaH virulence gene. SEQ ID NOs:4-6 aresequences of oligonucleotides used as downstreams primers foramplification of the ipaH virulence gene. SEQ ID NO:7 is the sequence ofan oligonucleotide used as an upstream bumper for SDA amplification. SEQID NO:8 is the sequence of an oligonucleotide used as a downstreambumper for SDA amplification. SEQ ID NOs:9-10 are sequences of detectoroligonucleotides (probes or reporters) for the ipaH virulence gene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to oligonucleotides, amplification primersand assay probes which exhibit Shigella and EIEC specificity in nucleicacid amplification reactions. Also provided are methods for detectingand identifying Shigella spp.s' and EIECs' nucleic acids using theoligonucleotides of the invention. The preferred methods are to use SDA,tSDA or homogeneous real time fluorescent tSDA. These methods are knownto those skilled in the art from references such as U.S. Pat. No.5,547,861, U.S. Pat. No. 5,648,211, U.S. Pat. No. 5,846,726 and U.S.patent application Ser. No. 08/865,675, filed May 30, 1997, thedisclosures of which are hereby specifically incorporated herein byreference. The use of microelectronic arrays for the analysis of nucleicacids are known to those skilled in the art from references such as U.S.Pat. No. 5,605,662 and U.S. Pat. No. 5,632,957 and PCT publishedapplication Nos. WO 96/01836 and WO 97/12030.

The primers of the present invention were designed based on an analysisof the ipaH virulence gene of Shigella spp. and EIEC to identifyShigella- and EIEC-specific regions. Primers developed for use in SDAare shown in Table 1. Also shown are probes for the detection of theresultant amplicons. The exemplary restriction endonuclease recognitionsites (BsoBI) in the amplification primers are shown in boldface typeand the target binding sequences are italicized. The target bindingsequence of an amplification primer determines its target specificity.

TABLE 1 Amplification Oligonucleotides        Upstream Primers ShH1AL48:5′-CGATTCCGCTCCAGACTTCTCGGG TCAGAAGCCGTGAAGA (SEQ ID 1) ShH1AL46:5′-CGATTCCGCTCCAGACTTCTCGGG TCAGAAGCCGTGAAG (SEQ ID 2) ShH1AL44:5′-CGATTCCGCTCCAGACTTCTCGGG CAGAAGCCGTGAAG (SEQ ID 3)        DownstreamPrimers ShH1AR50: 5′-ACCGCATCGAAGTCATGTCTCGGG GCCATGGTCCCCAGA (SEQ ID 4)ShH1AR46: 5′-ACCGCATCGAAGTCATGTCTCGGG CATGGTCCCCAGAG (SEQ ID 5)ShH1AR42: 5′-ACCGCATCGAAGTCATGTCTCGGG CATGGTCCCCAGA (SEQ ID 6)       Upstream Bumper ShH1BL44: 5′-GCACTGCCGAAGC (SEQ ID 7)        DownstreamBumper ShH1BBR44: 5′-GCTTCAGTACAGCAT (SEQ ID 8)        Detector ProbesSh1DL44: 5′-GAATTTACGGACTGG (SEQ ID 9) Sh1DR46: 5′-GAACCAGTCCGTAAAT (SEQID 10)

As nucleic acids do not require complete complementarity in order tohybridize, it is to be understood that the probe and primer sequencesherein disclosed may be modified to some extent without loss of utilityas Shigella- and EIEC-specific probes and primers. As is known in theart, hybridization of complementary and partially complementary nucleicacid sequences may be obtained by adjustment of the hybridizationconditions to increase or decrease stringency (i.e., adjustment ofhybridization pH, temperature or salt content of the buffer). Such minormodifications of the disclosed sequences and any necessary adjustmentsof hybridization conditions to maintain Shigella- and EIEC-specificityrequire only routine experimentation and are within the ordinary skillin the art.

The amplification products generated using the primers disclosed hereinmay be detected by a characteristic size, for example, on polyacrylamideor agarose gels stained with ethidium bromide. Alternatively, amplifiedtarget sequences may be detected by means of an assay probe, which is anoligonucleotide tagged with a detectable label. In one embodiment, atleast one tagged assay probe may be used for detection of amplifiedtarget sequences by hybridization (a detector probe), by hybridizationand extension as described by Walker, et al. (1992, Nucl. Acids Res.20:1691-1696) (a detector primer) or by hybridization, extension andconversion to double stranded form as described in EP 0 678 582 (asignal primer). SEQ ID NO:9 and SEQ ID NO:10 are particularly useful asdetector primers, i.e., primer extension detector probes, in conjunctionwith the amplification primers of the invention for detection ofShigella spp. and EIEC. Preferably, the assay probe is selected tohybridize to a sequence in the target that is between the amplificationprimers, i.e., it should be an internal assay probe. Alternatively, anamplification primer or the target binding sequence thereof may be usedas the assay probe.

The detectable label of the assay probe is a moiety which can bedetected either directly or indirectly as an indication of the presenceof the target nucleic acid. For direct detection of the label, assayprobes may be tagged with a radioisotope and detected by autoradiographyor tagged with a fluorescent moiety and detected by fluorescence as isknown in the art. Alternatively, the assay probes may be indirectlydetected by tagging with a label that requires additional reagents torender it detectable. Indirectly detectable labels include, for example,chemiluminescent agents, enzymes that produce visible reaction productsand ligands (e.g., haptens, antibodies or antigens) which may bedetected by binding to labeled specific binding partners (e.g.,antibodies or antigens/haptens). Ligands are also useful forimmobilizing the ligand-labeled oligonucleotide (the capture probe) on asolid phase to facilitate its detection. Particularly useful labelsinclude biotin (detectable by binding to labeled avidin or streptavidin)and enzymes such as horseradish peroxidase or alkaline phosphatase(detectable by addition of enzyme substrates to produce colored reactionproducts). Methods for adding such labels to, or including such labelsin, oligonucleotides are well known in the art and any of these methodsare suitable for use in the present invention.

Examples of specific detection methods which may be employed include achemiluminescent method in which amplified products are detected using abiotinylated capture probe and an enzyme-conjugated detector probe asdescribed in U.S. Pat. No. 5,470,723. After hybridization of these twoassay probes to different sites in the assay region of the targetsequence (between the binding sites of the two amplification primers),the complex is captured on a streptavidin-coated microtiter plate bymeans of the capture probe, and the chemiluminescent signal is developedand read in a luminometer. As another alternative for detection ofamplification products, a signal primer as described in EP 0 678 582 maybe included in the SDA reaction. In this embodiment, labeled secondaryamplification products are generated during SDA in a targetamplification-dependent manner and may be detected as an indication oftarget amplification by means of the associated label.

For commercial convenience, amplification primers for specific detectionand identification of nucleic acids may be packaged in the form of akit. Typically, such a kit contains at least one pair of amplificationprimers. Reagents for performing a nucleic acid amplification reactionmay also be included with the target-specific amplification primers, forexample, buffers, additional primers, nucleotide triphosphates, enzymes,etc. The components of the kit are packaged together in a commoncontainer, optionally including instructions for performing a specificembodiment of the inventive methods. Other optional components may alsobe included in the kit, e.g., an oligonucleotide tagged with a labelsuitable for use as an assay probe, and/or reagents or means fordetecting the label.

The target binding sequences of the amplification primers confer specieshybridization specificity on the oligonucleotides and therefore providespecies specificity to the amplification reaction. Thus, the targetbinding sequences of the amplification primers of the invention are alsouseful in other nucleic acid amplification protocols such as the PCR,conventional SDA (a reaction scheme which is essentially the same asthat of tSDA but conducted at lower temperatures using mesophilicenzymes), 3SR, NASBA and TAS. Specifically, any amplification protocolwhich utilizes cyclic, specific hybridization of primers to the targetsequence, extension of the primers using the target sequence as atemplate and separation or displacement of the extension products fromthe target sequence may employ the target binding sequences of theinvention. For amplification methods that do not require specialized,non-target binding sequences (e.g., PCR), the amplification primers mayconsist only of the target binding sequences of the amplificationprimers listed in Table 1.

Other sequences, as required for performance of a selected amplificationreaction, may optionally be added to the target binding sequencesdisclosed herein without altering the species specificity of theoligonucleotide. By way of example, the specific amplification primersmay contain a recognition site for the restriction endonuclease BsoBIwhich is nicked during the SDA reaction. It will be apparent to oneskilled in the art that other nickable restriction endonucleaserecognition sites may be substituted for the BsoBI recognition siteincluding, but not limited to, those recognition sites disclosed in EP 0684 315. Preferably, the recognition site is for a thermophilicrestriction endonuclease so that the amplification reaction may beperformed under the conditions of tSDA. Similarly, the tail sequence ofthe amplification primer (5′ to the restriction endonuclease recognitionsite) is generally not critical, although the restriction site used forSDA and sequences which will hybridize either to their own targetbinding sequence or to the other primers should be avoided. Someamplification primers for SDA therefore consist of 3′ target bindingsequences, a nickable restriction endonuclease recognition site 5′ tothe target binding sequence and a tail sequence about 10-25 nucleotidesin length 5′ to the restriction endonuclease recognition site. Thenickable restriction endonuclease recognition site and the tail sequenceare sequences required for the SDA reaction. For other amplificationreactions (e.g., 3SR, NASBA and TAS), the amplification primers mayconsist of the target binding sequence and additional sequences requiredfor the selected amplification reaction (e.g., sequences required forSDA as described above or a promoter recognized by RNA polymerase for3SR). Adaptation of the target binding sequences of the invention toamplification methods other than SDA employs routine methods forpreparation of amplification primers, such as chemical synthesis, andthe well known structural requirements for the primers of the selectedamplification reaction. The target binding sequences of the inventionmay therefore be readily adapted to Shigella- and EIEC-specific targetamplification and detection in a variety of amplification reactionsusing only routine methods for production, screening and optimization.

In SDA, the bumper primers are not essential for species specificity, asthey function to displace the downstream, species-specific amplificationprimers. It is required only that the bumper primers hybridize to thetarget upstream from the amplification primers so that when they areextended they will displace the amplification primer and its extensionproduct. The particular sequence of the bumper primer is thereforegenerally not critical, and may be derived from any upstream targetsequence which is sufficiently close to the binding site of theamplification primer to allow displacement of the amplification primerextension product upon extension of the bumper primer. Occasionalmismatches with the target in the bumper primer sequence or somecross-hybridization with non-target sequences do not generallynegatively affect amplification efficiency as long as the bumper primerremains capable of hybridizing to the specific target sequence.

Amplification reactions employing the primers of the invention mayincorporate thymine as taught by Walker, et al. (1992, Nucl. Acids Res.20:1691-1696), or they may wholly or partially substitute2′-deoxyuridine 5′-triphosphate for TTP in the reaction to reducecross-contamination of subsequent amplification reactions, e.g., astaught in EP 0 624 643. dU (uridine) is incorporated into amplificationproducts and can be excised by treatment with uracil DNA glycosylase(UDG). These abasic sites render the amplification product unamplifiablein subsequent amplification reactions. UDG may be inactivated by uracilDNA glycosylase inhibitor (UGI) prior to performing the subsequentamplification to prevent excision of dU in newly-formed amplificationproducts.

SDA is an isothermal method of nucleic acid amplification in whichextension of primers, nicking of a hemimodified restriction endonucleaserecognition/cleavage site, displacement of single stranded extensionproducts, annealing of primers to the extension products (or theoriginal target sequence) and subsequent extension of the primers occursconcurrently in the reaction mix. This is in contrast to PCR, in whichthe steps of the reaction occur in discrete phases or cycles as a resultof the temperature cycling characteristics of the reaction. SDA is basedupon 1) the ability of a restriction endonuclease to nick the unmodifiedstrand of a hemiphosphorothioate form of its double strandedrecognition/cleavage site and 2) the ability of certain polymerases toinitiate replication at the nick and displace the downstreamnon-template strand. After an initial incubation at increasedtemperature (about 95° C.) to denature double stranded target sequencesfor annealing of the primers, subsequent polymerization and displacementof newly synthesized strands takes place at a constant temperature.Production of each new copy of the target sequence consists of fivesteps: 1) binding of amplification primers to an original targetsequence or a displaced single-stranded extension product previouslypolymerized, 2) extension of the primers by a 5′-3′ exonucleasedeficient polymerase incorporating an α-thio deoxynucleosidetriphosphate (α-thio dNTP), 3) nicking of a hemimodified double strandedrestriction site, 4) dissociation of the restriction enzyme from thenick site, and 5) extension from the 3′ end of the nick by the 5′-3′exonuclease deficient polymerase with displacement of the downstreamnewly synthesized strand. Nicking, polymerization and displacement occurconcurrently and continuously at a constant temperature becauseextension from the nick regenerates another nickable restriction site.When a pair of amplification primers is used, each of which hybridizesto one of the two strands of a double stranded target sequence,amplification is exponential. This is because the sense and antisensestrands serve as templates for the opposite primer in subsequent roundsof amplification. When a single amplification primer is used,amplification is linear because only one strand serves as a template forprimer extension. Examples of restriction endonucleases which nick theirdouble stranded recognition/cleavage sites when an α-thio dNTP isincorporated are HincII, HindII, AvaI, NciI and Fnu4HI. All of theserestriction endonucleases and others which display the required nickingactivity are suitable for use in conventional SDA. However, they arerelatively thermolabile and lose activity above about 40° C.

Targets for amplification by SDA may be prepared by fragmenting largernucleic acids by restriction with an endonuclease which does not cut thetarget sequence. However, it is generally preferred that target nucleicacids having selected restriction endonuclease recognition/cleavagesites for nicking in the SDA reaction be generated as described byWalker, et al. (1992, Nucl. Acids Res. 20:1691-1696) and in U.S. Pat.No. 5,270,184 (herein incorporated by reference). Briefly, if the targetsequence is double stranded, four primers are hybridized to it. Two ofthe primers (S₁ and S₂) are SDA amplification primers and two (B₁ andB₂) are external or bumper primers. S₁ and S₂ bind to opposite strandsof double stranded nucleic acids flanking the target sequence. B₁ and B₂bind to the target sequence 5′ (i.e., upstream) of S₁ and S₂,respectively. The exonuclease deficient polymerase is then used tosimultaneously extend all four primers in the presence of threedeoxynucleoside triphosphates and at least one modified deoxynucleosidetriphosphate (e.g., 2′-deoxyadenosine 5′-O-(1-thiotriphosphate),“dATPαS”). The extension products of S₁ and S₂ are thereby displacedfrom the original target sequence template by extension of B₁ and B₂.The displaced, single stranded extension products of the amplificationprimers serve as targets for binding of the opposite amplification andbumper primer (e.g., the extension product of S₁ binds S₂ and B₂). Thenext cycle of extension and displacement results in two double strandednucleic acid fragments with hemimodified restriction endonucleaserecognition/cleavage sites at each end. These are suitable substratesfor amplification by SDA. As in SDA, the individual steps of the targetgeneration reaction occur concurrently and continuously, generatingtarget sequences with the recognition/cleavage sequences at the endsrequired for nicking by the restriction enzyme in SDA. As all of thecomponents of the SDA reaction are already present in the targetgeneration reaction, target sequences generated automatically andcontinuously enter the SDA cycle and are amplified.

To prevent cross-contamination of one SDA reaction by the amplificationproducts of another, dUTP may be incorporated into SDA-amplified DNA inplace of dTTP without inhibition of the amplification reaction. Theuracil-modified nucleic acids may then be specifically recognized andinactivated by treatment with uracil DNA glycosylase (UDG). Therefore,if dUTP is incorporated into SDA-amplified DNA in a prior reaction, anysubsequent SDA reactions can be treated with UDG prior to amplificationof double stranded targets, and any dU containing DNA from previouslyamplified reactions will be rendered unamplifiable. The target DNA to beamplified in the subsequent reaction does not contain dU and will not beaffected by the UDG treatment. UDG may then be inhibited by treatmentwith UGI prior to amplification of the target. Alternatively, UDG may beheat-inactivated. In tSDA, the higher temperature of the reaction itself(≧50° C.) can be used concurrently to inactivate UDG and amplify thetarget.

SDA requires a polymerase which lacks 5′-3′ exonuclease activity,initiates polymerization at a single stranded nick in double strandednucleic acids, and displaces the strand downstream of the nick whilegenerating a new complementary strand using the unnicked strand as atemplate. The polymerase must extend by adding nucleotides to a free3′-OH. To optimize the SDA reaction, it is also desirable that thepolymerase be highly processive to maximize the length of targetsequence which can be amplified. Highly processive polymerases arecapable of polymerizing new strands of significant length beforedissociating and terminating synthesis of the extension product.Displacement activity is essential to the amplification reaction, as itmakes the target available for synthesis of additional copies andgenerates the single stranded extension product to which a secondamplification primer may hybridize in exponential amplificationreactions. Nicking activity of the restriction enzyme is also of greatimportance, as it is nicking which perpetuates the reaction and allowssubsequent rounds of target amplification to initiate.

tSDA is performed essentially as the conventional SDA described byWalker, et al. (1992, Proc. Natl. Acad. Sci. USA 89:392-396 and 1992,Nucl. Acids Res. 20:1691-1696), with substitution of the desiredthermostable polymerase and thermostable restriction endonuclease. Ofcourse, the temperature of the reaction will be adjusted to the highertemperature suitable for the substituted enzymes and the HincIIrestriction endonuclease recognition/cleavage site will be replaced bythe appropriate restriction endonuclease recognition/cleavage site forthe selected thermostable endonuclease. Also in contrast to Walker, etal., the practitioner may include the enzymes in the reaction mixtureprior to the initial denaturation step if they are sufficiently stableat the denaturation temperature. Preferred restriction endonucleases foruse in thermophilic SDA are BsrI, BstNI, BsmAI, BslI and BsoBI (NewEngland BioLabs), and BstOI (Promega). The preferred thermophilicpolymerases are Bca (Panvera) and Bst (New England Biolabs).

Homogeneous real time fluorescent tSDA is a modification of tSDA. Itemploys detector oligonucleotides to produce reduced fluorescencequenching in a target-dependent manner. The detector oligonucleotidescontain a donor/acceptor dye pair linked such that fluorescencequenching occurs in the absence of target. Unfolding or linearization ofan intramolecularly base-paired secondary structure in the detectoroligonucleotide in the presence of the target increases the distancebetween the dyes and reduces fluorescence quenching. Unfolding of thebase-paired secondary structure typically involves intermolecularbase-pairing between the sequence of the secondary structure and acomplementary strand such that the secondary structure is at leastpartially disrupted. It may be fully linearized in the presence of acomplementary strand of sufficient length. In a preferred embodiment, arestriction endonuclease recognition site (RERS) is present between thetwo dyes such that intermolecular base-pairing between the secondarystructure and a complementary strand also renders the RERSdouble-stranded and cleavable or nickable by a restriction endonuclease.Cleavage or nicking by the restriction endonuclease separates the donorand acceptor dyes onto separate nucleic acid fragments, furthercontributing to decreased quenching. In either embodiment, an associatedchange in a fluorescence parameter (e.g., an increase in donorfluorescence intensity, a decrease in acceptor fluorescence intensity ora ratio of fluorescence before and after unfolding) is monitored as anindication of the presence of the target sequence. Monitoring a changein donor fluorescence intensity is preferred, as this change istypically larger than the change in acceptor fluorescence intensity.Other fluorescence parameters such as a change in fluorescence lifetimemay also be monitored.

A detector oligonucleotide for homogeneous real time fluorescent tSDA isan oligonucleotide which comprises both a single-stranded 5′ or 3′section which hybridizes to the target sequence (the target bindingsequence), as well as an intramolecularly base-paired secondarystructure adjacent to the target binding sequence. The detectoroligonucleotides of the invention further comprise a donor/acceptor dyepair linked to the detector oligonucleotide such that donor fluorescenceis quenched when the secondary structure is intramolecularly base-pairedand unfolding or linearization of the secondary structure results in adecrease in fluorescence quenching. Cleavage of an oligonucleotiderefers to breaking the phosphodiester bonds of both strands of a DNAduplex or breaking the phosphodiester bond of single-stranded DNA. Thisis in contrast to nicking, which refers to breaking the phosphodiesterbond of only one of the two strands in a DNA duplex.

The detector oligonucleotides of the invention for homogeneous real timefluorescent tSDA comprise a sequence which forms an intramolecularlybase-paired secondary structure under the selected reaction conditionsfor primer extension or hybridization. The secondary structure ispositioned adjacent to the target binding sequence of the detectoroligonucleotide so that at least a portion of the target bindingsequence forms a single-stranded 3′ or 5′ tail. As used herein, the term“adjacent to the target binding sequence” means that all or part of thetarget binding sequence is left single-stranded in a 5′ or 3′ tail whichis available for hybridization to the target. That is, the secondarystructure does not comprise the entire target binding sequence. Aportion of the target binding sequence may be involved in theintramolecular base-pairing in the secondary structure, it may includeall or part of a first sequence involved in intramolecular base-pairingin the secondary structure, it may include all or part of a firstsequence involved in intramolecular base-pairing in the secondarystructure but preferably does not extend into its complementarysequence. For example, if the secondary structure is a stem-loopstructure (e.g., a “hairpin”) and the target binding sequence of thedetector oligonucleotide is present as a single-stranded 3′ tail, thetarget binding sequence may also extend through all or part of the firstarm of the stem and, optionally, through all or part of the loop.However, the target binding sequence preferably does not extend into thesecond arm of the sequence involved in stem intramolecular base-pairing.That is, it is desirable to avoid having both sequences involved inintramolecular base-pairing in a secondary structure capable ofhybridizing to the target. Mismatches in the intramolecularlybase-paired portion of the detector oligonucleotide secondary structuremay reduce the magnitude of the change in fluorescence in the presenceof target but are acceptable if assay sensitivity is not a concern.Mismatches in the target binding sequence of the single-stranded tailare also acceptable but may similarly reduce assay sensitivity and/orspecificity. However, it is a feature of the present invention thatperfect base-pairing in both the secondary structure and the targetbinding sequence do not compromise the reaction. Perfect matches in thesequences involved in hybridization improve assay specificity withoutnegative effects on reaction kinetics.

When added to the amplification reaction, the detector oligonucleotidesignal primers of the invention are converted to double-stranded form byhybridization and extension as described above. Strand displacement bythe polymerase also unfolds or linearizes the secondary structure andconverts it to double-stranded from by synthesis of a complementarystrand. The RERS, if present, also becomes double-stranded and cleavableor nickable by the restriction endonuclease. As the secondary structureis unfolded or linearized by the strand displacing activity of thepolymerase, the distance between the donor and acceptor dye isincreased, thereby reducing quenching of donor fluorescence. Theassociated change in fluorescence of either the donor or acceptor dyemay be monitored or detected as an indication of amplification of thetarget sequence. Cleavage or nicking of the RERS generally furtherincreases the magnitude of the change in fluorescence by producing twoseparate fragments of the double-stranded secondary amplificationproduct, each having one of the two dyes linked to it. These fragmentsare free to diffuse in the reaction solution, further increasing thedistance between the dyes of the donor/acceptor pair. An increase indonor fluorescence intensity or a decrease in acceptor fluorescenceintensity may be detected and/or monitored as an indication that targetamplification is occurring or has occurred, but other fluorescenceparameters which are affected by the proximity of the donor/acceptor dyepair may also be monitored. A change in fluorescence intensity of thedonor or acceptor may also be detected as a change in a ratio of donorand/or acceptor fluorescence intensities. For example, a change influorescence intensity may be detected as; a) an increase in the ratioof donor fluorophore fluorescence after linearizing or unfolding thesecondary structure and donor fluorophore fluorescence in the detectoroligonucleotide prior to linearizing or unfolding, or b) as a decreasein the ratio of acceptor dye fluorescence after linearizing or unfoldingand acceptor dye fluorescence in the detector oligonucleotide prior tolinearizing or unfolding.

It will be apparent that, in addition to SDA, the detectoroligonucleotides of the invention may be adapted for use as signalprimers in other primer extension amplification methods (e.g., PCR, 3SR,TMA or NASBA). For example, the methods may be adapted for use in PCR byusing PCR amplification primers and a strand displacing DNA polymerasewhich lacks 5′→3′ exonuclease activity (e.g., Sequencing Grade Taq fromPromega or exo⁻ Vent or exo⁻ Deep Vent from New England BioLabs) in thePCR. The detector oligonucleotide signal primers hybridize to the targetdownstream from the PCR amplification primers, are displaced and arerendered double-stranded essentially as described for SDA. In PCR anyRERS may optionally be selected for use in the detector oligonucleotide,as there are typically no modified deoxynucleoside triphosphates presentwhich might induce nicking rather than cleavage of the RERS. Asthermocycling is a feature of amplification by PCR, the restrictionendonuclease is preferably added at low temperature after the finalcycle of primer annealing and extension for end-point detection ofamplification. However, a thermophilic restriction endonuclease thatremains active through the high temperature phases of the PCR reactioncould be present during amplification to provide a real-time assay. Asin SDA systems, separation of the dye pair reduces fluorescencequenching, with a change in a fluorescence parameter such as intensityserving as an indication of target amplification.

The change in fluorescence resulting from unfolding or linearizing ofthe detector oligonucleotides may be detected at a selected endpoint inthe reaction. However, because linearized secondary structures areproduced concurrently with hybridization or primer extension, the changein fluorescence may also be monitored as the reaction is occurring,i.e., in “real-time”. This homogeneous, real-time assay format may beused to provide semiquantitative or quantitative information about theinitial amount of target present. For example, the rate at whichfluorescence intensity changes during the unfolding or linearizingreaction (either as part of target amplification or in non-amplificationdetection methods) is an indication of initial target levels. As aresult, when more initial copies of the target sequence are present,donor fluorescence more rapidly reaches a selected threshold value(i.e., shorter time to positivity). The decrease in acceptorfluorescence similarly exhibits a shorter time to positivity, detectedas the time required to reach a selected minimum value. In addition, therate of change in fluorescence parameters during the course of thereaction is more rapid in samples containing higher initial amounts oftarget than in samples containing lower initial amounts of target (i.e.,increased slope of the fluorescence curve). These or other measurementsas is known in the art may be made as an indication of the presence oftarget or as an indication of target amplification. The initial amountof target is typically determined by comparison of the experimentalresults to results for known amounts of target.

Assays for the presence of a selected target sequence according to themethods of the invention may be performed in solution or on a solidphase. Real-time or endpoint homogeneous assays in which the detectoroligonucleotide functions as a primer are typically performed insolution. Hybridization assays using the detector oligonucleotides ofthe invention may also be performed in solution (e.g., as homogeneousreal-time assays) but are also particularly well-suited to solid phaseassays for real-time or endpoint detection of target. In a solid phaseassay, detector oligonucleotides may be immobilized on the solid phase(e.g., beads, membranes or the reaction vessel) via internal or terminallabels using methods known in the art. For example, a biotin-labeleddetector oligonucleotide may be immobilized on an avidin-modified solidphase where it will produce a change in fluorescence when exposed to thetarget under appropriate hybridization conditions. Capture of the targetin this manner facilitates separation of the target from the sample andallows removal of substances in the sample that may interfere withdetection of the signal or other aspects of the assay. An example of asolid phase system that can be used is an electronic microarray, i.e.,an active programmable electronic matrix hybridization system.

A simplified version of the active programmable electronic matrixhybridization system for use with this invention is described asfollows. Generally, a substrate supports a matrix or array ofelectronically addressable microlocations. A permeation layer isdisposed above the individual electrodes. The permeation layer permitstransport of relatively small charged entities through it, but precludeslarge charged entities, such as DNA from contacting the electrodesdirectly. The permeation layer avoids the electrochemical degradationthat would occur in the DNA by direct contact with the electrodes. Itfurther serves to avoid the strong, non-specific adsorption of DNA toelectrodes. Attachment regions are disposed upon the permeation layerand provide for specific binding sites for target materials.

In operation, a reservoir comprises that space above the attachmentregions that contains the desired (as well as undesired) materials fordetection, analysis or use. Charged entities, such as charged DNA, arelocated within the reservoir. In one aspect, the active, programmablematrix system comprises a method for transporting the charged materialto any of the specific microlocations. When activated, a microlocationgenerates the free field electrophoretic transport of any charged,functionalized, specific binding entity towards the electrode. Forexample, if one electrode were made positive and a second electrodenegative, electrophoretic lines of force would run between twoelectrodes. The lines of electrophoretic force cause transport ofcharged binding entities that have a net negative charge toward thepositive electrode. Charged materials having a net positive charge moveunder the electrophoretic force toward the negatively charged electrode.When the net negatively charged binding entity that has beenfunctionalized contacts the attachment layer as a result of its movementunder electrophoretic force, the finctionalized specific binding entitybecomes covalently attached to the attachment layer corresponding to thefirst electrode.

Electrophoretic transport generally results from applying a voltage,which is sufficient to permit electrolysis and ion transport within thesystem. Electrophoretic mobility results, and current flows through thesystem, such as by ion transport through the electrolyte solution. Inthis way, a complete circuit may be formed via the current flow of theions, with the remainder of the circuit being completed by theconventional electronic components, such as the electrodes andcontrolled circuitry. For example, for an aqueous electrolyte solutioncontaining conventional material such as sodium chloride, sodiumphosphate, buffers and ionic species, the voltage which induceselectrolysis and ion transport is greater than, or equal to,approximately 1.2 volts.

It is possible to protect the attachment layers that are not subject toreaction by making their corresponding electrodes negative. This resultsin electrophoretic lines of force emanating from such attachmentregions. The electrophoretic force lines serve to drive away negativelycharged binding entities from the non-reactant attachment layer andtowards the attachment layer corresponding to the first electrode. Inthis way, “force field” protection is formed around the attachmentlayers that it is desired to have nonreactive with the charged moleculesat that time.

One highly advantageous result of this system is that charged bindingmaterials may be highly concentrated in regions adjacent to the signalattachment layers. For example, if an individual microlocation ispositively charged, and the remaining microlocations are negativelycharged, the lines of electrophoretic force will cause transport of thenet negatively charged binding entities toward the positively chargedmicrolocation. In this way, a method for concentrating and reactinganalytes or reactants at any specific microlocation on the device may beachieved. After the attachment of the specific binding entities to theattachment layer, the underlying microelectrode may continue to fimctionin a direct current (DC) mode. This unique feature allows relativelydilute charged analytes or reactant molecules free in solution to betransported rapidly, concentrated, and reacted in a serial or parallelmanner at any specific microlocation that is maintained at the oppositecharge to the analyte or reactant molecules. This ability to concentratedilute analyte or reactant molecules at selected microlocations greatlyaccelerates the reaction rates at these microlocations.

After the desired reaction is complete, the electrode may have itspotential reversed, thereby creating an electrophoretic force in thedirection opposite the prior attractive force. In this way, nonspecificanalytes or unreacted molecules may be removed from the microlocation.Specific analytes or reaction products may be released from anymicrolocation and transported to other locations for further analysis,stored at other addressable locations, or removed completely from thesystem. This removal or deconcentration of materials by reversal of thefield enhances the discrimination ability of the system by resulting inremoval of nonspecifically bound materials. By controlling the amount ofnow-repulsive electrophoretic force to nonspecifically bound materialson the attachment layer, electronic stringency control may be achieved.By raising the electric potential at the electrode so as to create afield sufficient to remove partially hybridized DNA sequences, therebypermitting identification of single mismatched hybridizations, pointmutations may be identified.

Operations may be conducted in parallel or in series at the variousattachment layers. For example, a reaction may occur first at a firstattachment layer, utilizing the potentials as shown. The potential at afirst electrode may be reversed, that is, made negative, and thepotential at the adjacent second electrode may be made positive. In thisway, a series reaction occurs. Materials that were not specificallybound to the first attachment layer would be transported byelectrophoretic force to the attachment layer. In this way, theconcentration aspect is utilized to provide high concentrations at thatspecific attachment layer then subject to the positive electrophoreticforce. The concentrated materials may next be moved to an adjacent, orother, attachment layer. Alternatively, multiple attachment layers maybe deprotected in the sense that there is a net electrophoretic forcefield emanating from the electrode through the attachment layer out intothe reservoir. By deprotecting the multiple attachment layer, multiplexreactions are performed. Each individual site may serve in essence as aseparate biological “test tube” in that the particular environmentaddressed by a given attachment layer may differ from those environmentssurrounding the other attachment layers.

In one embodiment, the permeation layer contains avidin and one of theSDA primers contains biotin. Subsequent to amplification, the ampliconsare electronically addressed onto the array and binds to the avidin. Oneor more labeled detector probes are then added and allowed to hybridizewith the amplicons. The presence of hybridized detector probes is thendetected. In a second embodiment, one or more capture probes aredesigned to hybridize with the amplified nucleic acid. Each captureprobe contains biotin and is either bound onto or electronicallyaddressed and bound onto an array in which the permeation layer containsavidin. The amplicons are then electronically addressed onto the arrayand hybridize with the capture probes. One or more labeled detectorprobes are then added and allowed to hybridize with the amplicons. Thepresence of hybridized detector probes is then detected.

Further details of the electronic microarray and associated systems aredescribed by Heller et al. (1997, U.S. Pat. No. 5,605,662; 1997, U.S.Pat. No. 5,682,957; 1997, PCT published application No. WO97/12030), andSosnowski et al. (1998, PCT published application No. WO98/10273), thedisclosures of which are hereby specifically incorporated herein byreference.

In addition, techniques utilizing SDA and electronic microarrays,including several assay formats, are disclosed in copending applicationSer. No. 09/290,632, filed concurrently herewith, incorporated herein byreference. In one embodiment, described in this application, a sandwichassay is used in which a single-stranded capture probe is electronicallydeposited on the array, and serves to capture one strand of a chargedmolecule such as target nucleic acid or amplicon thereof. A multiplicityof molecules such as nucleic acid capture probes can be electronicallydeposited on different pads of the array. It is preferred that thehybridization of the target molecule or amplicon and the capture probebe conductd electronically. Following capture of the charged molecule tothe capture sites, the captured molecule may be detected by a labeledreporter probe that binds to the captured molecule.

In a second embodiment described in this application, an electronicamplification is conducted on the microarray. In this embodiment, targetnucleic acid is electronically concentrated in the vicinity of anchoredprimers located on a capture site and used in an SDA or otheramplification method. Electronic hybridization is used to hybridize thetemplate molecules to the anchored SDA primers. The microchips are thenincubated with an SDA reaction mix which contains the SDA componentsother than the template and the amplification primers. After thereaction is stopped, the products are denatured, and the microchipincubated with reporter probes to detect the presence of target nucleicacid. These embodiments illustrate that (a) the amplification may beconducted on an electronic microarray followed by analysis or (b) theamplification may be conducted in solution and then analysis conductedon an electronic microarray.

EXAMPLES

The following Examples illustrate specific embodiments of the inventiondescribed herein. As would be apparent to skilled artisans, variouschanges and modifications are possible, and are contemplated within thescope of the invention described.

Example 1 Primer Screening

All pairwise combinations of upstream and downstream amplificationprimers shown in Table I were tested for amplification of the target.Amplification reactions were conducted in the presence of 0, 10³ or 10⁶genomic equivalents of Shigella DNA. Amplification was performed at 52°C. in buffers containing final concentrations of the followingcomponents: 30-40 mM potassium phosphate (pH 7.6), 5-9% glycerol, 3-7%dimethylsulfoxide (DMSO), 5 mM magnesium acetate, 700 ng human placentalDNA, 10 μg acetylated bovine serum albumin, 1.82% trehalose, 0.36 mMdithiothreitol, 500 nM tSDA primers, 50 nM bumper primers, 0.25 mM dUTP,0.7 mM 2′-deoxycytidine 5′-O-(1-thiotriphosphate), 0.1 mM dATP, 0.1 mMdGTP, and approximately 640 units BsoBI and 40 units Bst polymerase.

In brief, target DNA was denatured for 5 minutes at 95° C. and cooled toroom temperature prior to addition to buffer containing the primers andbumpers. Incubation was continued at room temperature for 20 minutes,followed by incubation at 70° C. for 10 minutes to minimize potentialfalse priming. Amplification was then initiated at 52° C. by transfer ofa fixed volume of the priming mix to microtiter wells containing theamplification enzymes. Amplification was carried out for 1 hour at aconstant temperature of 52° C. Amplification products were detected byautoradiography following primer extension with ³²P-labeled detectorsequence SEQ ID NO:10 and resolution on 8% denaturing polyacrylamidegels. Specific amplification products were detected with the followingcombinations of amplification primers: SEQ ID NO:1 and SEQ ID NO:4, SEQID NO:1 and SEQ ID NO:5, SEQ ID NO:1 and SEQ ID NO:6, SEQ ID NO:2 andSEQ ID NO:5, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:3 and SEQ ID NO:5,and SEQ ID NO:3 and SEQ ID NO:6.

Example 2 Determination of Analytical Sensitivity

SDA was performed as described in Example 1 using amplification primersSEQ ID NO:1 and SEQ ID NO:5 and bumper primers SEQ ID NO:7 and SEQ IDNO: 8 in buffer containing 30 mM potassium phosphate, 9% glycerol and 3%DMSO. Target DNA was included at 0, 10², 10³ or 10⁴ genomic equivalentsper reaction. An analytical sensitivity of 10² was demonstrated throughthe detection of specific amplification products in reactions containingthis level of input target.

Example 3 Evaluation of Primer Specificity

Primer specificity was evaluated using SEQ ID NO:1, SEQ ID NO:5, SEQ IDNO:7 and SEQ ID NO:8 as described in Example 1 with SDA buffercontaining final concentrations of 30 mM potassium phosphate, 9%glycerol and 3% DMSO. Seventy-two strains of Shigella representing allfour species within the genus and seven strains of EIEC were tested at10⁴-10⁵ genomic equivalents per reaction (Table 2). Seventy-one strainsof Shigella yielded specific product as did all seven strains of EIECfor a calculated specificity of 99%.

TABLE 2 Shigella/EIEC Specificity Panel Species Strain 1 S. boydii ATCC12027 2 S. boydii ATCC 12031 3 S. boydii ATCC 12032* 4 S. boydii ATCC12034 5 S. boydii ATCC 12035 6 S. boydii ATCC 25930 7 S. boydii ATCC35964 8 S. boydii ATCC 35965 9 S. boydii ATCC 35966 10 S. boydii ATCC49812 11 S. boydii ATCC 8702 12 S. boydii ATCC 8704 13 S. boydii ATCC9207 14 S. boydii ATCC 9210 15 S. boydii ATCC 9905 16 S. dysenteriaeATCC 9361 17 S. dysenteriae ATCC 9750 18 S. dysenteriae BDMS 2896 19 S.dysenteriae BDMS 2932 20 S. dysenteriae BDMS 2933 21 S. dysenteriae BDMS2943 22 S. dysenteriae BDMS 2947 23 S. dysenteriae BDMS 3197 24 S.dysenteriae BDMS 3198 25 S. dysenteriae BDMS 3205 26 S. dysenteriae BDMS9945 27 S. dysenteriae BDMS 9946 28 S. flexneri ATCC 11836 29 S.flexneri ATCC 12022 30 S. flexneri ATCC 12023 31 S. flexneri ATCC 1202532 S. flexneri ATCC 49070 33 S. flexneri ATCC 9199 34 S. flexneri ATCC9204 35 S. flexneri ATCC 9380 36 S. flexneri ATCC 9473 37 S. flexneriATCC 9748 38 S. flexneri ATCC 9906 39 S. flexneri JHU 17628 40 S.flexneri JHU 21431 41 S. flexneri JHU 24929 42 S. flexneri JHU 24997 43S. flexneri VTU 982-6065 44 S. flexneri VTU 982-6068 45 S. flexneri VTU982-6103 46 S. flexneri VTU 982-6208 47 S. flexneri VTU 982-6250 48 S.sonnei ATCC 25931 49 S. sonnei ATCC 29029 50 S. sonnei ATCC 9290 51 S.sonnei BDMS 2888 52 S. sonnei BDMS 2894 53 S. sonnei BDMS 2928 54 S.sonnei BDMS 2948 55 S. sonnei BDMS 2949 56 S. sonnei BDMS 4424 57 S.sonnei BDMS 5701 58 S. sonnei BDMS 5702 59 S. sonnei BDMS 5704 60 S.sonnei BDMS 7140 61 S. sonnei BDMS 8154 62 S. sonnei BDMS 8996 63 S.sonnei BDMS 9108 64 S. sonnei BDMS 9490 65 S. sonnei BDMS 9921 66 S.sonnei VTU 982-6211 67 S. sonnei VTU 982-6227 68 S. sonnei VTU 982-624469 S. sonnei VTU 982-6246 70 S. sonnei VTU 982-6247 71 S. sonnei VTU982-6259 72 S. sonnei VTU 982-6262 73 E. coli 0124:B17:H ATCC 12806 74E. coli 0124 L-73C-7 75 E. coli 028 D-233 76 E. coli 0144:H25 EI-1109 77E. coli 029:H EI-1150 78 E. coli 0136 CW-139-8 79 E. coli 0136 U-346*Negative by SDA for ipaH target sequence

Example 4 Evaluation of Cross-Reactivity

Cross-reactivity was evaluated using the primers and reaction conditionsdescribed in Example 3. Non-Shigella/EIEC organisms were tested at 10⁷genomic equivalents per reaction. Negative results were obtained withall 25 organisms tested (Table 3). In each case, when 10⁴ copies ofShigella target DNA were seeded into the reaction, specific product wasobtained indicating that the non-Shigella/EIEC DNA did not inhibitamplification.

TABLE 3 Shigella/EIEC Cross-Reactivity Panel Species Strain 1Actinomyces israelii Serotype 1 ATCC 10049 2 Arcobacter butzleri ATCC49616 3 Arcobacter cryaerophilus ATCC 49942 4 Bacteroides ureolyticusATCC 33387 5 Campylobacter coli ATCC 49941 6 Campylobacter jejuni ATCC49943 7 Campylobacter lari ATCC 43675 8 Candida albicans ATCC 44808 9Citrobacter freundii ATCC 8090 10 Enterobacter cloacae ATCC 13047 11Enterococcus faecalis ATCC 29212 12 Escherichia coli 055 ATCC 12014 13Escherichia coli 0157:H7 non-SLTI T-3785 14 Escherichia coli T-4025 15Helicobacter pylori ATCC 43526 16 Klebsiella pneumoniae subsp.pneumoniae ATCC 13883 17 Proteus mirabilis ATCC 29906 18 Salmonellaenteritidis ATCC 13076 19 Salmonella minnesota ATCC 9700 20 Salmonellatyphimurium ATCC 13311 21 Staphylococcus aureus subsp aureus ATCC 1259822 Streptococcus bovis ATCC 9809 23 Streptococcus pyogenes Group A ATCC19615 24 Vibrio cholerae Biotype El Tor ATCC 14035 25 Yersiniaenterocolitica ATCC 9610

Example 5 Electronic Microarray Analysis

The microelectronic array assembly has been described previously (R. G.Sosnowski et al., 1997, Proc. Natl. Acad. Sci. USA 94:119-123).Electronic targeting of capture probes, amplicons or detector probesutilized conditions reported elsewhere (R. G. Sosnowski et al., 1997,Proc. Natl. Acad Sci. USA 94:119-123; C. F. Edman et al., 1997, NucleicAcids Res. 25:4907-4914). The permeation layer of the microelectronicarray assembly advantageously contains avidin. In brief, capture probesare electronically addressd onto a microelectronic array. Crudeamplification reactions are either spun for 2 min through G6 columns(Biorad, Hercules, Calif.) preequilibrated with distilled water ordialyzed in multiwell plates (Millipore, Bedford, Mass.) for ≧5 hrsagainst distilled water. The prepared samples are then mixed in a 1:1ratio with 100 mM histidine and heated at 95° C. for 5 min prior toelectronic addressing. For detection, a fluorescent labeledoligonucleotide (detector probe) is introduced in 6×SSC and allowed tohybridize for 30 min at room temperature. The array is then washed in0.1×STE/1%SDS followed by 1×STE. The presence of detector probe is thendetected.

While the invention has been described with some specificity,modifications apparent to those of ordinary skill in the art may be madewithout departing from the scope of the invention. Various features ofthe invention are set forth in the following claims.

10 1 40 DNA Artificial Sequence Description of Artificial SequencePrimerfor SDA of Shigella spp./EIEC 1 cgattccgct ccagacttct cgggtcagaagccgtgaaga 40 2 39 DNA Artificial Sequence Description of ArtificialSequencePrimer for SDA of Shigella spp./EIEC 2 cgattccgct ccagacttctcgggtcagaa gccgtgaag 39 3 38 DNA Artificial Sequence Description ofArtificial SequencePrimer for SDA of Shigella spp./EIEC 3 cgattccgctccagacttct cgggcagaag ccgtgaag 38 4 39 DNA Artificial SequenceDescription of Artificial SequencePrimer for SDA of Shigella spp./EIEC 4accgcatcga agtcatgtct cggggccatg gtccccaga 39 5 38 DNA ArtificialSequence Description of Artificial SequencePrimer for SDA of Shigellaspp./EIEC 5 accgcatcga agtcatgtct cgggcatggt ccccagag 38 6 37 DNAArtificial Sequence Description of Artificial SequencePrimer for SDA ofShigella spp./EIEC 6 accgcatcga agtcatgtct cgggcatggt ccccaga 37 7 13DNA Artificial Sequence Description of Artificial SequenceBumper primerfor SDA of Shigella spp./EIEC 7 gcactgccga agc 13 8 15 DNA ArtificialSequence Description of Artificial SequenceBumper primer for SDA ofShigella spp./EIEC 8 gcttcagtac agcat 15 9 15 DNA Artificial SequenceDescription of Artificial SequenceDetecto r probe for Shigella spp./EIEC9 gaatttacgg actgg 15 10 16 DNA Artificial Sequence Description ofArtificial SequenceDetecto r probe for Shigella spp./EIEC 10 gaaccagtccgtaaat 16

What is claimed is:
 1. An oligonucleotide consisting of a target bindingsequence selected from the group consisting of the target bindingsequences of ShH1AL48 (SEQ ID NO:1), ShH1AL46 (SEQ ID NO:2), SbH1AL44(SEQ ID NO:3), of ShH1AR50 (SEQ ID NO:4), ShH1AR46 (SEQ ID NO:5) andShH1AR42 (SEQ ID NO:6), and optionally, a sequence required for anamplification reaction.
 2. The oligonucleotide of claim 1 wherein thesequence required for the amplification reaction is a restrictionendonuclease recognition site which is nicked by a restrictionendonuclease during Strand Displacement Amplification.
 3. Theoligonucleotide of claim 2 selected from the group consisting ofShH1AL48 (SEQ ID NO:1), ShH1AL46 (SEQ ID NO:2), ShH1AL44 (SEQ ID NO:3),of ShH1AR50 (SEQ ID NO:4), ShH1AR46 (SEQ ID NO:5) and ShH1AR42 (SEQ IDNO:6).
 4. An oligonucleotide consisting of ShH1BL44 (SEQ ID NO:7) orShH1BBR44 (SEQ ID NO:8).
 5. An oligonucleotide selected from the groupconsisting of Sh1DL44 (SEQ ID NO:9), a nucleic acid complementary to SEQID NO:9, Sh1DR46 (SEQ ID NO:10) and a nucleic acid complementary to SEQID NO:
 10. 6. The nucleic acid of claim 5 wherein said nucleic acidcomprises a detectable marker.
 7. The nucleic acid of claim 6 whereinsaid detectable marker is selected from the group consisting of aradioactive marker and a fluorescence marker.
 8. A pair of amplificationprimers comprising: a) a first primer consisting of a target bindingsequence selected from the group consisting of the target bindingsequences of ShH1AL48 (SEQ ID NO:1), ShH1AL46 (SEQ ID NO:2), ShH1AL44(SEQ ID NO:3), and, optionally, a sequence required for an amplificationreaction, and; b) a second primer consisting of a target bindingsequence selected from the group consisting of the target bindingsequences of ShH1AR50 (SEQ ID NO:4), ShH1AR46 (SEQ ID NO:5) and ShH1AR42(SEQ ID NO:6), and, optionally, a sequence required for an amplificationreaction.
 9. The pair of amplification primers of claim 8 wherein thesequence required for the amplification reaction is a restrictionendonuclease recognition site which is nicked by a restrictionendonuclease during Strand Displacement Amplification.
 10. The pair ofamplification primers of claim 9 wherein said first primer is selectedfrom the group consisting of ShH1AL48 (SEQ ID NO:1), SbH1AL46 (SEQ IDNO:2), ShH1AL44 (SEQ ID NO:3) and said second primer is selected fromthe group consisting of ShH1AR50 (SEQ ID NO:4), ShH1AR46 (SEQ ID NO:5)and ShH1AR42 (SEQ ID NO:6).
 11. The pair of amplification primers ofclaim 9 wherein said first primer is ShH1AL48 (SEQ ID NO:1) and saidsecond primer is ShH1AR46 (SEQ ID NO:5).